Microorganism for converting carbon dioxide to aliphatic carboxylic acids via formic acid intermediate

ABSTRACT

The invention relates to a micro-organism comprising a hydrogenase enzyme system which is capable of converting carbon dioxide into formic acid and a second enzyme system which is capable of converting formic acid into aliphatic carboxylic acids having a chain length of five or more carbon atoms. Also described are various methods for producing oil, as well as other aspects of the invention.

FIELD OF THE INVENTION

The present invention relates to a micro-organism which is capable ofconverting carbon dioxide to formic acid which is then converted intoaliphatic carboxylic acids of longer chain length. Particular enzymesystems in the micro-organism are responsible for these reactions.Further aspects of the invention relate to methods of producing formicacid and the aliphatic carboxylic acids, and the aliphatic carboxylicacids themselves.

BACKGROUND TO THE INVENTION

Over recent years, there has been increasing concern over theconsumption of fossil fuels and the production of greenhouse gases. Oneway to reduce the globe's reliance on fossil fuels has been thedevelopment of biofuels from renewable sources. Biofuels such asbiodiesel and bioethanol are considered to be cleaner and moreenvironmentally friendly alternatives to fossil fuels.

Although biofuels may help in reducing green house emissions, they arenot without problems. A controversial aspect is the “food for fuel”problem where the demand for energy crops has been perceived as pushingup the prices of grain commodities. Another serious drawback is thedamage caused to ecologically sensitive ecosystems, such as rainforests, where the planting of energy crops such as soya and palm hascaused large scale destruction.

The biofuels industry is turning to second and third generation biofuelsto alleviate these issues. The production of fuels by micro-organisms(1) and the use of waste substrates (2) are important areas of research.

The conversion of carbon dioxide to fuel molecules is known. Carbondioxide can be converted chemically (3), electrochemically (4), andeither directly (5) or indirectly (6) by micro-organisms. Products suchas formic acid, formate, methanol, formaldehyde, ethylene, methane andoxalic acid have been noted. However, these micro-organisms cannotconvert carbon dioxide via formic acid into a longer chain energy sourcesuch as aliphatic carboxylic acids.

In US 2012003705, the conversion of carbon dioxide to biomass isdescribed (7) and then the further processing of the biomass to a rangeof commercially useful molecules. However, this is not done through thesteps of fixing carbon dioxide to formic acid and then converting theformic acid to aliphatic carboxylic acids.

Previous attempts at using carbon dioxide as a carbon substrate toproduce fuel molecules have had limitations. Carbon dioxide and itsaqueous ions bicarbonate and carbonate are inherently stable and theGibbs free energy of formation are the most electronegative of thecarbon molecules. To convert carbon dioxide to fuel molecules requires alarge input of energy (heat), extreme conditions (pressure) and highlyreactive chemicals (catalysts). Yields are often poor and the rates ofreaction slow. Chemical approaches to the direct use of carbon dioxideare generally not considered to be economically viable. Likewise, theinitial production of biomass by chemolithotrophic bacteria is notwidely practised due to cost constraints of growth and downstreamprocessing.

Electro-catalysis has also had limited success. Outcomes have beenlimited by the poor solubility of carbon dioxide in water (0.033M) andthe energetic requirements of a reaction with a strong electronegativepotential (EO=−0.61V). Electro-catalysis is also a costly technologyrequiring high quality metals for electrode surfaces. Production oflonger chain products have been described in terms of Fischer Tropschtype reactions (8), but again chains are limited in length.Photoreduction on irradiated semi-conductor surfaces produces carbonmonoxide, formate, methanol, methane, formaldehyde, oxalic acid andglyoxal. Again this is a costly technology with low yields.

While enzymes such as bacterial formate dehydrogenase are known toreduce carbon dioxide to formate (9), the forward reaction (oxidation offormate to carbon dioxide) is generally favoured because NADPH isrequired to drive the reaction and the reduction potential of NADP ismore positive than that of carbon dioxide. Such a reaction also requireselectron donor and acceptor molecules. Tungsten containing enzymes fromSyntrophotobacterium fumioxidans (10) are able to carry out thisreaction but require absorption onto an electrode surface for theelectro-catalytic system to function efficiently. Further, longer fuelmolecules are not produced.

The present invention is an improvement compared to the prior art inthat carbon dioxide can be converted to an initial platform molecule(formic acid) and then assembled into longer chains in a rapid formatthat does not require fermentation or the generation and processing ofbiomass. This improves on known methods such as US2012/0003705 (11),which requires the production of biomass and recycling of electrondonors and acceptors, and US 2010/03170741A1 (12), US 2012/0003706A1(13), US2012/003707A (14) and US2012/0034664A1 (15) which all requirefermentative processes.

SUMMARY OF THE INVENTION

A new micro-organism has been isolated by the inventor of thisapplication which is capable of fixing carbon dioxide and converting itinto an aliphatic carboxylic acid energy source. Therefore, in general,the invention relates to such a micro-organism comprising a hydrogenaseenzyme system which can convert carbon dioxide into formic acid. Themicro-organism also comprises a second enzyme system which can convertformic acid into aliphatic carboxylic acids having a chain length offive or more carbon atoms. This means that the carboxylic acids that canbe produced have five or more carbon atoms in total in the compound.Additionally, the second enzyme system can also produce aliphaticcarboxylic acids having a chain length of two, three and four carbonatoms. These shorter chain carboxylic acids are converted to aliphaticcarboxylic acids having a chain length of five or more carbon atoms.

The micro-organism isolated by the inventor, an Acetobacter lovaniensis,is suitable for the production of oils on a commercial basis. Thismicro-organism uses carbon dioxide as its sole source of carbon andproduces carboxylic acids of various lengths. The fact that themicro-organism uses carbon dioxide as its sole carbon source means thatoil production is much more affordable than for other micro-organismswhich have to be supplied with a hydrocarbon substrate to perform thesame task (16, 17, 18). The use of such a micro-organism to convertcarbon dioxide to a combustible fuel is of commercial value. Further,since the micro-organism does not require a hydrocarbon substrate, theneed for the production of energy crops is removed.

The invention improves on current methods in that carbon dioxide isconverted to a liquid fuel product without the need for fermentation orproduction and processing of biomass. Additionally, the conversion ofcarbon dioxide to fuel molecules is a very attractive option in that isboth a free source of renewable carbon and its sequestration haspositive effects for the environment.

Further advantages associated with the isolated micro-organism are that:

1) it is a non-pathogenic organism and is rated Class 1;

2) it does not require special growth conditions or large growth volumesas would be required for algae;

3) it has a robust hydrogenase enzyme system;

4) oil is synthesised external to the cell and is therefore easier toharvest and adapt to a commercial manufacturing process;

5) the oil produced consists mostly of long chain carboxylic acids. Thisoil can be used directly as a source of energy by combustion, or used asa feed stock in a number of industries such as in the production ofbiodiesel, detergents and various oleo chemicals;

6) the enzyme system will function in oil-water emulsions which aredescribed as a novel route of manufacture in that the oil in theemulsion acts as a primer for chain building and increases thesolubility of carbon dioxide in the reaction media; and

7) the enzyme system operates without the need to produce and processbiomass.

Other aspects of the invention relate to methods of producing formicacid and the aliphatic carboxylic acid energy source, and the energysource itself.

These and further aspects of the invention will be described in moredetail below.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect of the invention, there is provided a micro-organismcomprising a hydrogenase enzyme system which is capable of convertingcarbon dioxide into formic acid and a second enzyme system which iscapable of converting formic acid into aliphatic carboxylic acids havinga chain length of five or more carbon atoms.

The micro-organism may be any suitable micro-organism as long as it iscapable of converting carbon dioxide into formic acid and thenconverting the formic acid into aliphatic carboxylic acids having achain length of five or more carbon atoms. Preferably, themicro-organism is a prokaryote. More preferably, the micro-organism is abacterium. In one embodiment, the micro-organism is lithotrophic. Alithotroph is an organism that uses an inorganic substrate (usually ofmineral origin) to obtain reducing equivalents for use in biosynthesis(e.g., carbon dioxide fixation) or energy conservation via aerobic oranaerobic respiration. The micro-organism may be lithoautotrophic. Alithoautotroph is able to use carbon dioxide from the air as a carbonsource. The micro-organism may use carbon dioxide as the sole source ofcarbon. The micro-organism may be chemolithotrophic. Chemolithotrophsuse inorganic compounds for aerobic or anaerobic respiration. The energyproduced by the oxidation of these compounds is enough for ATPproduction. Some of the electrons derived from the inorganic donors alsoneed to be channeled into biosynthesis. In one embodiment, themicro-organism is chemolithoautotrophic.

The terms “lithotrophic”, “lithoautotrophic”, “chemolithotrophic” and“chemolithoautotrophic” used above are well known to those skilled inthe art and have a precise meaning which is widely recognised. As aresult, a skilled person would readily be able to determine whether aparticular micro-organism, such as a bacterium, falls within thedefinition of one or more of these terms. Further, a skilled personcould also test any micro-organisms of interest to determine whetherthey were classed in one or more of these categories. A skilled personcould also test a micro-organism to see if it can produce an oil ofaliphatic carboxylic acids having a chain length of five or more carbonatoms. Suitable methods for conducting such tests are well known tothose skilled in the art. In one embodiment, the micro-organism is anaerobic micro-organism. Again, it is well within the capabilities of aperson skilled in the art to determine whether a particularmicro-organism is an aerobic micro-organism. In the context of thepresent invention, the micro-organism, and more particularly themicro-organism's enzyme systems, produce aliphatic carboxylic acidsunder aerobic conditions, i.e. the presence of oxygen is tolerated inthe reactions that produce the aliphatic carboxylic acids.

Where the micro-organism is a bacterium, the micro-organism may be anysuitable bacterium which comprises a hydrogenase enzyme system capableof converting carbon dioxide to formic acid and a second enzyme systemwhich is capable of converting formic acid into aliphatic carboxylicacids having a chain length of five or more carbon atoms. Preferably,the bacterium is an Acetobacter species. In one particular embodiment,the micro-organism is Acetobacter lovaniensis. The micro-organism may besimilar to the Acetobacter strain having accession number NCIMB 41808(deposited at NCIMB Ltd. (Ferguson Building, Craibstone Estate,Bucksburn, Aberdeen, AB21 9YA) on 12 Jan. 2011 under the provisions ofthe Budapest Treaty; hereinafter referred to as strain FJ1). The term“similar to” means a micro-organism which is functionally equivalent toFJ1. The micro-organism should have a hydrogenase enzyme system which iscapable of converting carbon dioxide into formic acid and should be ableto grow under the same conditions as FJ1. Further, the micro-organismshould comprise the same or similar enzymatic pathways for producingaliphatic carboxylic acids having a chain length of five or more carbonatoms. The micro-organism may have at least about 60% sequence identityto FJ1. In some embodiments, the micro-organism may have at least about65%, at least about 70%, at least about 75% or at least about 80%sequence identity to FJ1. Preferably, the micro-organism should have atleast about 85%, at least about 90%, at least about 93%, at least about95%, at least about 97%, at least about 98%, or at least about 99%sequence identity to FJ1. Methods for determining sequence identitybetween different micro-organisms are well known to those skilled in theart. For example, 16S rDNA analysis can be used. Detailed informationregarding the culturing of FJ1 is given below. Therefore, it is wellwithin the capabilities of a person skilled in the art to determinewhether a micro-organism is similar to FJ1. In one embodiment, themicro-organism is FJ1.

In one particular embodiment, the micro-organism may be a recombinantmicro-organism, i.e., a genetically engineered micro-organism. Themicro-organism may comprise a nucleotide sequence (e.g. DNA) fromanother species of micro-organism. In particular, the micro-organism maycomprise a heterologous gene or genes encoding the hydrogenase enzymesystem. The heterologous gene or genes may be operatively linked to aheterologous promoter or to a promoter of the micro-organism. Theheterologous gene or genes may be part of a plasmid (22). Theheterologous gene or genes will be expressed in the micro-organism toproduce a functional hydrogenase enzyme system allowing themicro-organism to convert carbon dioxide to formic acid. Suitablemethods for introducing nucleotide sequences (e.g. DNA) of interest intomicro-organism host cells are well known to those skilled in the art(For example, see Sambrook, J. and Russell, D. Molecular Cloning: ALaboratory Manual. Cold Spring Harbor Laboratory Press, U.S).

Alternatively, the micro-organism may be a naturally occurringmicro-organism. This is a micro-organism which has not been geneticallyaltered or modified.

In one embodiment, the micro-organism may be derived from FJ1. The term“derived from” means that FJ1 can be modified or mutated to producefurther micro-organisms according to the invention. For example, genesmay be inserted or removed from FJ1. Micro-organisms which are derivedfrom FJ1 should be functionally equivalent to FJ1 and should have ahydrogenase enzyme system which is capable of converting carbon dioxideinto formic acid. Further, the derived micro-organisms should be able togrow under the same conditions as FJ1. Furthermore, the derivedmicro-organisms should comprise the same or similar enzymatic pathwaysfor producing aliphatic carboxylic acids having a chain length of fiveor more carbon atoms. The derived micro-organisms can produce aliphaticcarboxylic acids in the same way as FJ1. In some embodiments,micro-organisms may be derived by repeatedly culturing and selectingmicro-organisms in an artificial selection process.

The hydrogenase enzyme system can be any suitable enzyme system which iscapable of converting carbon dioxide into formic acid. The hydrogenaseenzyme system catalyses the conversion of carbon dioxide to formic acid.The hydrogenase enzyme system oxidises hydrogen to generate theelectrons to reduce carbon dioxide to formic acid, for example, usingthe following reaction:

CO₂+H₂→HCOOH

Generally, catalysis of the conversion of carbon dioxide to formic acidtakes place in solution as carbon dioxide is soluble. In solution,carbon dioxide is reversibly converted to carbonic acid (H₂CO₃).Depending on the pH of the solution, the carbonic acid will normally bepresent as bicarbonate (HCO₃ ⁻) or carbonate (CO₃ ²⁻). Therefore, theconversion of carbon dioxide to formic acid by the hydrogenase enzymesystem of the present invention also encompasses the conversion ofbicarbonate and/or carbonate to formic acid. In other words, carbonateand bicarbonate are considered to be forms of carbon dioxide in thepresent invention. Bicarbonate and/or carbonate salts (such as sodiumbicarbonate or sodium carbonate) may be added to solution to increasethe level of these salts. However, this is not preferred as the saltions (e.g. sodium ions) may form soap when the aliphatic carboxylicacids are produced.

As indicated above, carbon dioxide is preferably the sole source ofcarbon. Preferably, the carbon dioxide is converted directly from carbondioxide to formic acid without the formation of any stableintermediates. In other words, the reaction occurs in a single step.Carbonate and bicarbonate are to be considered as forms of carbondioxide which are in solution and are not intermediates. Therefore, thedissolution of carbon dioxide to carbonate and bicarbonate ions insolution and then to formic acid is considered to be direct conversion.On the other hand, conversion of carbon dioxide (or carbonate and/orbicarbonate ions) to methanol and then to formic acid is not consideredto be direct conversion as a methanol intermediate is produced.

In some embodiments, the starting reactants for producing formic acidare water and carbon dioxide. In preferred embodiments, no otherreactants are necessary to produce formic acid other than carbon dioxideand water. This does not, however, rule out the possibility that othercomponents will be present in the initial reaction mixture. For example,an oxidising agent may be present to speed up the initiation of thereactions. Such other components are not reactants in the production offormic acid.

The hydrogenase enzyme system does not require electron acceptor anddonor molecules in order to produce formic acid. For example, moleculessuch as NADP and NADPH are not required for the reaction to proceed. Infact, all the reactions to produce the aliphatic carboxylic acids havinga chain length of five or more do not require electron acceptor or donormolecules.

The hydrogenase enzyme system does not use fermentation to produceformic acid. Further, neither does the second enzyme system usefermentation to produce the aliphatic carboxylic acids. Fermentation isthe conversion of organic compounds such as carbohydrates into othercompounds through biochemical processes involving electron acceptorsand/or donors.

The hydrogenase enzyme system does not require biomass to produce formicacid. Therefore, no biomass needs to be provided to the micro-organismin order for aliphatic carboxylic acids to be produced. Biomass isbiological organic material from plants or animals that can be convertedto an energy source.

In the present invention, an electrochemical process such aselectrocatalysis does not need to be used to convert carbon dioxide toformic acid and then to aliphatic carboxylic acids. An electrochemicalprocess is a chemical reaction which take place in a solution at theinterface of an electron conductor (a metal or a semiconductor) and anionic conductor (the electrolyte), and which involves electron transferbetween the electrode and the electrolyte or species in solution.

The hydrogenase enzyme system is preferably oxygen tolerant. This meansthat the hydrogenase enzyme system can tolerate relatively high levelsof oxygen without damaging the enzyme system or affecting the activityof the enzyme system. Preferably, the hydrogenase enzyme system canfunction at an oxygen level of more than about 10%, more preferably, atan oxygen level of more than about 15%, and even more preferably, at anoxygen level of between about 20% and about 21%, e.g. at the level ofoxygen found in the atmosphere (about 20.95%). Many hydrogenase enzymesare sensitive to the presence of oxygen and effectively stop workingwhen oxygen is present. Preferably, the hydrogenase enzyme system isextracellular, i.e. outside the cell of the micro-organism. In otherwords, the hydrogenase enzyme system is positioned outside the cellmembrane. It is orientated extracellularly. Preferably, the hydrogenaseenzyme system is completely extracellular so that it is not attached tothe micro-organism in any way, for example, by being attached to thecell membrane of the micro-organism. This advantageously allows formicacid formation to take place outside the cell of the micro-organism. Thehydrogenase enzyme system is preferably functional between pH 3.0 and8.5 and, more preferably, between pH 3.5 and 4.5. Further, thehydrogenase enzyme system is preferably functional between 5° C. and 60°C. and, more preferably, between 15° C. and 20° C.

The hydrogenase enzyme system may comprise one or more enzymes, inaddition to the hydrogenase enzyme, to aid in the conversion of carbondioxide to formic acid.

In one embodiment, the hydrogenase enzyme system is that of FJ1. Inanother aspect of the invention, there is provided the hydrogenaseenzyme system of FJ1.

The micro-organism also comprises a second enzyme system which iscapable of converting formic acid into aliphatic carboxylic acids havinga chain length of five or more carbon atoms. The second enzyme systemcan be any suitable enzyme system which is capable of converting formicacid to aliphatic carboxylic acids having a chain length of five or morecarbon atoms. The second enzyme system catalyses the conversion offormic acid to aliphatic carboxylic acids having a chain length of fiveor more carbon atoms. The aliphatic carboxylic acids can be used, e.g.in combustion, to produce energy.

Preferably, the second enzyme system is extracellular, i.e. outside thecell of the micro-organism. In other words, In other words, the secondenzyme system is positioned outside the cell membrane. The second enzymesystem is orientated extracellularly. This means that the conversion offormic acid to aliphatic carboxylic acids takes place extracellularly.This advantageously allows the aliphatic carboxylic acids to be easilyextracted once produced by the micro-organism. Preferably, the secondenzyme system is completely extracellular so that it is not attached tothe micro-organism in any way, for example, by being attached to thecell membrane of the micro-organism.

In one embodiment, the second enzyme system is that of FJ1. In anotheraspect of the invention, there is provided the second enzyme system ofFJ1.

As indicated above, the micro-organism may be a recombinantmicro-organism. Therefore, the micro-organism may comprise aheterologous gene or genes encoding the second enzyme system. Theheterologous gene or genes may be operatively linked to a heterologouspromoter or to a promoter of the micro-organism. The heterologous geneor genes may be part of a plasmid. The heterologous gene or genes willbe expressed in the micro-organism to produce a functional second enzymesystem allowing the micro-organism to convert formic acid into aliphaticcarboxylic acids having a chain length of five or more carbon atoms.

The precise nature of the aliphatic carboxylic acids that are producedby the micro-organism will depend, in part, on the length of time of theenzymatic reactions.

The aliphatic carboxylic acids produced by the micro-organism can have achain length of five or more carbon atoms. Aliphatic carboxylic acidshaving a chain length of two, three and four carbon atoms are alsoproduced by the micro-organism. The aliphatic carboxylic acids that areproduced may be fatty acids. The aliphatic carboxylic acids may be shortchain, medium chain or long chain aliphatic carboxylic acids, or acombination of these.

The term ‘aliphatic’ in the context of the aliphatic carboxylic acidsmeans that the group attached to the —COOH group of the carboxylic acidcomprises a chain of carbon atoms joined together to form the backboneof the group. This carbon backbone may be branched or unbranched.Preferably, the carbon backbone is unbranched. The carbon backbone maybe saturated, mono-unsaturated (i.e. one carbon-carbon double bond) orpoly-unsaturated (i.e. more than one carbon-carbon double bond). In oneembodiment, the carbon backbone is mono-unsaturated. The carbon backboneis generally bonded to hydrogen atoms (other than the —COOH group).However, instead of one or more of the hydrogen atoms, the carbonbackbone may be substituted with other groups such as OH groups.Preferably, the carbon backbone is unsubstituted, i.e. only hydrogenatoms are bonded to the carbon backbone other than the —COOH group.

The second enzyme system of the micro-organism which produces thealiphatic carboxylic acids from formic acid does so in a stepwisemanner. A carbon atom is added one at a time to the carbon backbone ofthe aliphatic carboxylic acids. This produces a range of carboxylicacids having a carbon backbone of C2, C3, C4, C5, C6, C7, C8, C9, C10,etc. For example, starting with formic acid (C1), the second enzymesystem can add a carbon atom to the carbon backbone of formic acid toproduce acetic acid (C2). Then, the second enzyme system can add acarbon atom to the carbon backbone of acetic acid to produce propionicacid (C3). This process can continue so that sequentially butyric acid(C4), valeric acid (C5), hexanoic acid (C6), heptanoic acid (C7),octanoic acid (C8), nonanoic acid (C9), decanoic acid (C10), etc. can beproduced. Therefore, the second enzyme system is capable of convertingformic acid into an aliphatic carboxylic acid having a chain length oftwo or more carbon atoms, three or more carbon atoms, four or morecarbon atoms, five or more carbon atoms, six or more carbon atoms, sevenor more carbon atoms, eight or more carbon atoms, nine or more carbonatoms, ten or more carbon atoms, etc. The second enzyme system cancatalyse the addition of a carbon atom (e.g. a CH₂ unit) to the carbonchain of aliphatic carboxylic acids.

In this way, the enzyme systems of the micro-organism can produce arange of different length aliphatic carboxylic acids. For example, themicro-organism may produce a range of aliphatic carboxylic acids havinga carbon backbone length of between about 2 and about 24 carbon atoms,between about 3 and about 24 carbon atoms, between about 4 and about 24carbon atoms or between about 5 and about 24 carbon atoms. Further, themicro-organism can produce a range of aliphatic carboxylic acids havinga carbon backbone length of between about 6 and about 24 carbon atoms,between about 7 and about 24 carbon atoms, between about 8 and about 24carbon atoms or between about 9 and about 24 carbon atoms. Additionally,the micro-organism can produce a range of aliphatic carboxylic acidshaving a carbon backbone length of between about 10 and about 24 carbonatoms, between about 11 and about 24 carbon atoms, between about 12 andabout 24 carbon atoms or between about 13 and about 24 carbon atoms.When the micro-organism is cultured, the length of the hydrocarbon chainis likely to be longer if the micro-organism is cultured for longersince the enzyme systems will have been active for longer. The periodfor which the micro-organism is cultured determines the average chainlength. In one embodiment, the aliphatic carboxylic acid contentcalculated as C18 is between about 70% and 90%. More preferably, thealiphatic carboxylic acid content calculated as C18 is about 80%.

Preferably, the aliphatic carboxylic acids produced by themicro-organism are combustible. Preferably, the aliphatic carboxylicacids produced by the micro-organism are in the form of an oil. Thismakes separation of the aliphatic carboxylic acids easier. The oil maybe a semi-drying oil. Whether an oil is semi-drying can be evaluatedfrom the iodine value. Standard methods of analysis are for example EN14111. Preferably, the iodine value of the oil is 85-95 mg 1/100 g. Morepreferably, the iodine value of the oil is 90-95 mg 1/100 g. Thealiphatic carboxylic acids may be mono-unsaturated.

An infrared scan of aliphatic carboxylic acids produced by amicro-organism of the invention can be seen in FIG. 1. This shows thatthe sample, which is an oil, is made up of long chain carboxylic acidsand is an aliphatic carboxylic acid.

The aliphatic carboxylic acids, once extracted, may have one or more ofthe following properties:

Analysis Typical Value Method of Analysis Ash g/100 g 0.025-0.050 ISO3987 Flash Point ° C. >100 EN ISO 3679 Kinematic Viscosity cSt EN ISO3104 @ 20° C. 70-75 @ 30° C. 52-57 @ 40° C. 47-52 @ 50° C. 30-35 @ 60°C. 20-25 @ 70° C. 15-20 @ 80° C. 10-15 @ 90° C.  7-12 Viscosity Index 55ASTM D2270 Water Content g/100 g <0.5 EN ISO 12937 Density kg/l @ 20° C.920-950 EN ISO 12185 Acid Value mg KOH/g 140-160 EN 14104 Iodine Valuemg I/100 g 85-95 EN 14111 Copper Strip Corrosion 1B ASTM D849 OxidativeStability H >48 hours ASTM D2274 Sulphur % m/m <0.1 (typically 0.05)ASTM D2622 Peroxide Value meq/kg <3 AOAC 965.33 Cetane Index ~50 ASTMD976 Gross Calorific Value >37 ASTM D5865 Mj/kg Sediment potential <0.2(typically 0.08) ASTM D-6469

In some embodiments, the kinematic viscosity (cSt) at 40° C. may be20-25. Further, the flask point may be more than 180° C.

It has been found that the oxidative stability of the aliphaticcarboxylic acids is surprisingly high compared to other biofuels such asbiodiesel. Normal biodiesel has an oxidative stability of about 30minutes (when tested with ASTMD2274). The aliphatic carboxylic acidshave an oxidative stability of more than 48 hours.

A typical composition for oils produced after different reaction lengthsis shown below. These tables show the boiling point of the variousfractions. So, when the aliphatic carboxylic acids are fractionated, thefractions are split up into bands and the boiling point of thesefractions measured. For example, the first 10% of the aliphaticcarboxylic acids when fractionated off will have a certain boilingpoint. The second 10% (i.e. 10-20% referred to below as 20%) will haveanother boiling point and so on.

Typical Distillation Range of a Heavier Fraction Produced by Reactionfor 1.5 Hours

Percentage Distillate Boiling Point ° C. Initial Boiling Point 188 10%232 20% 256 30% 272 40% 292 50% 306 60% 321 70% 332 80% 359 90% 391Final Boiling Point 395

Typical Distillation Range of a Lighter Fraction Produced by Reactionfor 0.5 Hours

Percentage Distillate Boiling Point ° C. Initial Boiling Point 99 10%113 20% 115 30% 118 40% 118 50% 119 60% 121 70% 129 80% 308 90% 370Final Boiling Point 395

As discussed above, the micro-organism can be produced recombinantly.Accordingly, in a further aspect of the invention, there is provided amethod of producing a micro-organism, the method comprising the step ofinserting a gene or genes encoding a hydrogenase enzyme system capableof converting carbon dioxide to formic acid into a micro-organism,wherein the gene or genes are expressed by the micro-organism.

The method further comprises the step of inserting a gene or genesencoding a second enzyme system which is capable of converting formicacid into aliphatic carboxylic acids having a chain length of five ormore carbon atoms, wherein the gene or genes are expressed by themicro-organism.

Also provided is a method of producing a micro-organism, the methodcomprising the step of inserting a gene or genes encoding a hydrogenaseenzyme system capable of converting carbon dioxide to formic acid into amicro-organism, wherein the micro-organism comprises a second enzymesystem which is capable of converting formic acid into aliphaticcarboxylic acids having a chain length of five or more carbon atoms, andwherein the gene or genes are expressed by the micro-organism.

Also provided is a method of producing a micro-organism, the methodcomprising the step of inserting a gene or genes encoding a secondenzyme system which is capable of converting formic acid into aliphaticcarboxylic acids having a chain length of five or more carbon atoms intoa micro-organism, wherein the micro-organism comprises a hydrogenaseenzyme system capable of converting carbon dioxide to formic acid, andwherein the gene or genes are expressed by the micro-organism.

The description above relating to the micro-organism of the invention,and the preferable features thereof, are equally applicable to themethods for producing the micro-organism. For example, the particularfeatures relating to the nature of the micro-organism are alsoapplicable to the methods.

In another aspect, the invention provides a method of producingaliphatic carboxylic acids, the method comprising culturing amicro-organism comprising a hydrogenase enzyme system which is capableof converting carbon dioxide to formic acid and a second enzyme systemwhich is capable of converting formic acid into aliphatic carboxylicacids having a chain length of five or more carbon atoms.

As indicated above, aliphatic carboxylic acids having a range of carbonbackbone lengths can be produced. Preferably, aliphatic carboxylic acidshaving a carbon backbone length of between about 5 and about 24 carbonatoms are produced. More preferably, aliphatic carboxylic acids having acarbon backbone length of between about 10 and about 24 carbon atoms areproduced. More preferably still, aliphatic carboxylic acids having acarbon backbone length of between about 16 and about 20 carbon atoms areproduced. In one embodiment, the aliphatic carboxylic acids have acarbon backbone length of more than 2, 3, 4, 5, 6, 7, 8, 9 or 10.Further, the aliphatic carboxylic acids may have a carbon backbonelength of less than 25.

Alternatively, the aliphatic carboxylic acid content calculated as C18is between about 70% and 90%. More preferably, the aliphatic carboxylicacid content calculated as C18 is about 80%.

Any suitable conditions may be used for culturing the micro-organism.The micro-organism may be cultured at a pH of between about 3.0 andabout 8.5 and, more preferably, between about 3.5 and about 4.5.Further, the micro-organism may be cultured at a temperature of betweenabout 5° C. and about 60° C. and, more preferably, between about 15° C.and about 20° C.

The culture solution will contain carbon dioxide. This can be carbondioxide which has dissolved from the air. Preferably, carbon dioxide gasis bubbled through the culture solution to increase the level of carbondioxide available to react. When carbon dioxide is bubbled through theculture solution, it can come from a compressed gas cylinder.Alternatively, it can be contained in gas from another source. Forexample, waste gases from combustion can be bubbled through the culturemedium.

In some embodiments, carbon dioxide (e.g. waste gases from combustion)can be bubbled through the culture solution (which may optionallycontain a carbon dioxide sequestering agent) before the addition of themicro-organism.

Preferably, the micro-organism is cultured with carbon dioxide as thesole source of carbon.

The culture medium in which the culturing of the micro-organism takesplace may be any suitable culture medium. Preferably, carbon dioxide isthe sole source of carbon in the medium (as mentioned above HCO₃ ⁻ andCO₃ ²⁻ are considered to be soluble forms of carbon dioxide and areencompassed by the term carbon dioxide. The culture medium may compriseone or more of the following components: KH₂PO₄; MgSO₄.7H₂O; CaCO₃;CuSO₄; FeCl₃; MnCl₃; MoCl₃; and ZnCl₃. Further, these components may bepresent in the culture medium at the following concentrations:

NUTRIENT g/litre KH₂PO₄ 1.00 MgSO₄•7H₂O 0.10 CaCO₃ 0.10 CuSO₄ 0.10 FeCl₃0.01 MnCl₃ 0.01 MoCl₃ 0.01 ZnCl₃ 0.01

Preferably, the micro-organism is cultured in solution in an oil-wateremulsion. This can be achieved by introducing some oil into the culturemedium and agitating the oil-water mixture so that it forms an emulsion.For example, circulation of the reaction mixture can be used. It isthought that using an oil-water emulsion helps to increase thesolubility of the carbon dioxide. It is also thought that this helps tokeep the aliphatic carboxylic acids in solution so that their chainlength can continue to increase.

Preferably, a carbon dioxide sequestering agent is introduced into thereaction mixture. This helps to increase the level of carbon dioxide inthe reaction mixture/solution in order to maintain/increase the rate ofreaction. Suitable carbon dioxide sequestering agents are well known tothose skilled in the art and include alkalai compounds such as sodiumhydroxide, potassium hydroxide, barium chloride, triethanolamine,diethanolamine, monoethanolamine, ammonia, methanol, sulfolane,polyethylene glycol ethers, polyethylene glycols, glycerol, andsurfactants such as the Triton TX series. Some suitable carbon dioxidesequestering agents are described in Ortrud Ashenbrenner and PeterStyring (2010) (21). Preferably, the carbon dioxide sequestering agentis triethanolamine or polyethylene glycol. In some embodiments, thecarbon dioxide sequestering agent is triethanolamine. In otherembodiments, the carbon dioxide sequestering agent is a polyethyleneglycol such as PEG 300. Advantageously, polyethylene glycols do notrequire a chemical reaction to release the carbon, dioxide, do notcontain nitrogen which can potentially form NOX emissions on combustion,do not form soaps, and help to reduce the ash in the final product.

Preferably, an oxidising agent is introduced into the reaction mixture.This helps initiate the reaction process and speeds up the start of thereaction. Suitable oxidising agents include sodium hypochlorite andsodium hydroxide. The oxidising agent should be a mild oxidising agent.In one embodiment, bleach can be used the oxidising agent.

The method may further comprise the step of separating the aliphaticcarboxylic acids.

In another aspect, the invention provides aliphatic carboxylic acidsproduced by the method described above.

In yet another aspect, the invention provides aliphatic carboxylic acidscreated from a series of condensation reactions between formic acidmolecules.

As indicated above, aliphatic carboxylic acids having a range of carbonbackbone lengths can be produced. Preferably, the aliphatic carboxylicacids have a carbon backbone length of between about 5 and about 24carbon atoms. More preferably, the aliphatic carboxylic acids have acarbon backbone length of between about 10 and about 24. More preferablystill, the aliphatic carboxylic acids have a carbon backbone length ofbetween about 16 and about 20 carbon atoms. Alternatively, the aliphaticcarboxylic acid content calculated as C18 is between about 70% and 90%.More preferably, the aliphatic carboxylic acid content calculated as C18is about 80%.

Preferably, the aliphatic carboxylic acids are in the form of an oil.This makes separation of the aliphatic carboxylic acids easier. The oilmay be a semi-drying oil. Preferably, the aliphatic carboxylic acids aremono-unsaturated.

The aliphatic carboxylic acids, once extracted, may have one or more ofthe following properties:

Analysis Typical Value Ash g/100 g 0.025-0.050 Density @ 15° C.0.8483-0.8720 Flash Point ° C. >100  Kinematic Viscosity cSt @ 20° C.70-75 @ 30° C. 52-57 @ 40° C. 47-52 @ 50° C. 30-35 @ 60° C. 20-25 @ 70°C. 15-20 @ 80° C. 10-15 @ 90° C.  7-12 Viscosity Index  55 Water Contentg/100 g 1 max (pref. <0.5) Acid Value mg KOH/g 140-160 Iodine Value mgI/100 g 85-95 Copper Strip Corrosion 1B Oxidative Stability H  48+Sulphur % m/m   <0.1 Peroxide Value meq/kg  <3 Cetane Index ~50 GrossCalorific Value >37 Mj/kg Sediment potential <0.2 (typically 0.08)

The invention also provides the use of the micro-organisms describedabove for producing aliphatic carboxylic acids.

In one embodiment, the enzymes responsible for converting carbon dioxideto formic acid and then to aliphatic carboxylic acids are extracellularof the micro-organism. These enzymes function regardless of whether thecells of the micro-organism are present. Therefore, in another aspect ofthe invention, there is provided a method for producing aliphaticcarboxylic acids, the method comprising producing aliphatic carboxylicacids using a medium comprising a hydrogenase enzyme system which iscapable of converting carbon dioxide into formic acid and a secondenzyme system which is capable of converting formic acid to aliphaticcarboxylic acids having a chain length of five or more carbon atoms.

The medium can be produced by culturing the micro-organism for a periodof time to allow the enzyme systems to be produced in the medium. Ifdesired, it is possible to produce a cell free extract which containsthe enzymes but not the cells of the micro-organism. This can be done,for example, by removing the cells of the micro-organism from the mediumafter culturing, for example, by repeated ultra-filtration.Alternatively, rather than removing the cells of the micro-organism fromthe medium, the cells can be left in the medium but instead killed, forexample, by introducing a disinfectant or antimicrobial agent into themedium. Alternatively, the micro-organisms can be left in the medium.

The method may further comprise preparing the medium before producingthe aliphatic carboxylic acids using the medium.

Any suitable conditions can be used for producing the aliphaticcarboxylic acids. For example, carbon dioxide and water are provided.The medium may have a pH of between about 3.0 and about 8.5 and, morepreferably, between about 6.0 and about 7.0. Further, the medium mayhave a temperature of between about 5° C. and about 60° C. and, morepreferably, between about 15° C. and about 20° C.

As with the first method, carbon dioxide is preferably used as the solesource of carbon, for example, by bubbling carbon dioxide gas throughthe reaction medium. Preferably, the reaction medium is an oil-wateremulsion. Further, a carbon dioxide sequestering agent is preferablyintroduced into the reaction medium.

The method may further comprise the step of separating the aliphaticcarboxylic acids.

In all embodiments of the aliphatic carboxylic acid production methodsdescribed above, once the aliphatic carboxylic acids have been produced,various optional steps may be carried out on the aliphatic carboxylicacids. For example, the methods may optionally comprise one or more ofthe following steps:

1) separating the aliphatic carboxylic acids;

2) filtering the aliphatic carboxylic acids;

3) blending the aliphatic carboxylic acids with a different fuel,preferably an oil fuel;

4) chemically modifying the carboxylic acids, for example, into ester,alcohols, ketones or aldehydes; and

5) distilling off certain fractions of the aliphatic carboxylic acids.

In another aspect, the invention provides aliphatic carboxylic acidsproduced by the methods described above.

One aspect of the invention also provides a medium comprising ahydrogenase enzyme system which is capable of converting carbon dioxideinto formic acid and a second enzyme system which is capable ofconverting formic acid to aliphatic carboxylic acids having a chainlength of five or more carbon atoms.

From the medium comprising a hydrogenase enzyme system which is capableof converting carbon dioxide into formic acid and a second enzyme systemwhich is capable of converting formic acid to aliphatic carboxylic acidshaving a chain length of five or more carbon atoms, it is possible toisolate the enzymes contained therein using various techniques. Forexample, the enzymes could be separated using known techniques such asgel exclusion chromatography and selected based on their molecular sizesand activities.

The invention also provides use of a hydrogenase enzyme system which iscapable of converting carbon dioxide into formic acid and a secondenzyme system which is capable of converting formic acid to aliphaticcarboxylic acids having a chain length of five or more carbon atoms forproducing aliphatic carboxylic acids.

Further, the invention provides a method of producing aliphaticcarboxylic acids, the method comprising converting carbon dioxide toformic acid and then converting formic acid to aliphatic carboxylicacids having a chain length of five or more carbon atoms, wherein theconversion reactions take place in an oil-water emulsion.

Preferred features of the above method are the same as for the method ofproducing aliphatic carboxylic acids above. For example, the featuresrelating to the range of carbon backbone lengths that are produced, thetemperature and pH at which the reactions are carried out, the carbondioxide source, the carbon dioxide sequestering agent, the oxidisingagent, etc. are preferred features for this method.

For example, the emulsion will contain carbon dioxide. This can becarbon dioxide which has dissolved from the air. Preferably, carbondioxide gas is bubbled through the emulsion to increase the level ofcarbon dioxide available to react. When carbon dioxide is bubbledthrough the emulsion, it can come from a compressed gas cylinder.Alternatively, it can be contained in gas from another source. Forexample, waste gases from combustion can be bubbled through theemulsion.

In some embodiments, carbon dioxide (e.g. waste gases from combustion)can be bubbled through the emulsion (which may optionally contain acarbon dioxide sequestering agent).

Preferably, carbon dioxide is the sole source of carbon.

Preferably, a carbon dioxide sequestering agent is introduced into theemulsion. This helps to increase the level of carbon dioxide in thereaction mixture/solution in order to maintain/increase the rate ofreaction. Suitable carbon dioxide sequestering agents are well known tothose skilled in the art and include alkali compounds such as sodiumhydroxide, potassium hydroxide, barium chloride, triethanolamine,diethanolamine, monoethanolamine, ammonia, methanol, sulfolane,polyethylene glycol ethers, polyethylene glycols, glycerol, andsurfactants such as the Triton TX series. Some suitable carbon dioxidesequestering agents are described in Ortrud Ashenbrenner and PeterStyring (2010) (21). Preferably, the carbon dioxide sequestering agentis triethanolamine or polyethylene glycol. In some embodiments, thecarbon dioxide sequestering agent is triethanolamine. In otherembodiments, the carbon dioxide sequestering agent is a polyethyleneglycol such as PEG 300. Advantageously, polyethylene glycols do notrequire a chemical reaction to release the carbon, dioxide, do notcontain nitrogen which can potentially form NOX emissions on combustion,do not form soaps, and help to reduce the ash in the final product.

Preferably, an oxidising agent is introduced into the emulsion. Thishelps initiate the reaction process and speeds up the start of thereaction. Suitable oxidising agents include sodium hypochlorite andsodium hydroxide. The oxidising agent should be a mild oxidising agent.In one embodiment, bleach can be used the oxidising agent.

The method may further comprise the step of separating the aliphaticcarboxylic acids or any one or more of the processing steps describedabove.

Additionally, the invention provides a method of producing aliphaticcarboxylic acids having a chain length of five or more carbon atoms, themethod comprising converting carbon dioxide to formic acid and thenconverting formic acid to aliphatic carboxylic acids having a chainlength of five or more carbon atoms, wherein an oxidising agent is usedto initiate the conversion of carbon dioxide to formic acid.

Preferred features of the above method are the same as for the methodsof producing aliphatic carboxylic acids above. For example, the featuresrelating to the range of carbon backbone lengths that are produced, thetemperature and pH at which the reactions are carried out, the carbondioxide source, the carbon dioxide sequestering agent, the emulsion,etc. are preferred features for this method.

For example, generally, the formic acid and aliphatic carboxylic acidswill be produced in solution. The solution will contain carbon dioxide.This can be carbon dioxide which has dissolved from the air. Preferably,carbon dioxide gas is bubbled through the solution to increase the levelof carbon dioxide available to react. When carbon dioxide is bubbledthrough the solution, it can come from a compressed gas cylinder.Alternatively, it can be contained in gas from another source. Forexample, waste gases from combustion can be bubbled through thesolution.

In some embodiments, carbon dioxide (e.g. waste gases from combustion)can be bubbled through the solution (which may optionally contain acarbon dioxide sequestering agent).

Preferably, carbon dioxide is the sole source of carbon.

Preferably, a carbon dioxide sequestering agent is introduced into thesolution. This helps to increase the level of carbon dioxide in thereaction mixture/solution in order to maintain/increase the rate ofreaction. Suitable carbon dioxide sequestering agents are well known tothose skilled in the art and include alkali compounds such as sodiumhydroxide, potassium hydroxide, barium chloride, triethanolamine,diethanolamine, monoethanolamine, ammonia, methanol, sulfolane,polyethylene glycol ethers, polyethylene glycols, glycerol, andsurfactants such as the Triton TX series. Some suitable carbon dioxidesequestering agents are described in Ortrud Ashenbrenner and PeterStyring (2010) (21). Preferably, the carbon dioxide sequestering agentis triethanolamine or polyethylene glycol. In some embodiments, thecarbon dioxide sequestering agent is triethanolamine. In otherembodiments, the carbon dioxide sequestering agent is a polyethyleneglycol such as PEG 300. Advantageously, polyethylene glycols do notrequire a chemical reaction to release the carbon, dioxide, do notcontain nitrogen which can potentially form NOX emissions on combustion,do not form soaps, and help to reduce the ash in the final product.

Preferably, the reactions take place in an oil-water emulsion.

The method may further comprise the step of separating the aliphaticcarboxylic acids or any one or more of the processing steps describedabove.

The method of producing oil is “driven” in a novel way. The first stepof the process is the reduction of carbon dioxide to produce formicacid. Formic acid is a reducing agent (redox potential −0.25). The firststep of the reaction fixes carbon dioxide but also generates sufficientreducing equivalents to drive the next part of the process (chainbuilding). The use of an “in situ” chemical battery could be used todrive other redox reactions. Formic acid (reductant) then reducesavailable carboxylic acids (oxidant), adding one carbon to a growingchain and releasing oxygen as O₂. This circumvents the need to add areducing agent to drive the reaction, carbon and reducing equivalentsbeing sequestered in the final product.

EXAMPLES

The invention will now be described in detail by way of example onlywith reference to the figures in which:

FIG. 1 is an infrared scan of an energy source produced by themicro-organism of the invention. The energy source is an oil productcomprising aliphatic carboxylic acids and the infrared scan showschemical features typical of an aliphatic carboxylic acid.

FIG. 2 shows a first tank system which can be used to produce oilaccording to the invention.

FIG. 3 shows a second tank system which can be used to produce oilaccording to the invention.

FIG. 4 is a flowchart showing the process for producing oil using thesecond tank system.

The following description is in no way limiting to the scope of theinvention.

Overview

The following aspects of the invention will be discussed:

1) Properties of the bacterial culture;

2) Production of carbon building blocks (formic acid);

3) Assembly of building blocks into short, medium and long chainaliphatic carboxylic acids; and

4) The product.

The Bacterial Culture

The bacterial culture preferably has the following properties:

-   -   It is a chemolithotrophic, aerobic organism growing on carbon        dioxide as its sole source of carbon.    -   It possesses an oxygen tolerant hydrogenase enzyme system that        is extracellular. This enzyme system should be stable and active        between pH 3.0 and 8.5 and between 5° C. and 60° C.    -   It possesses an extracellular enzyme component capable of        assembling formic acid into short, medium and long chain        hydrocarbon molecules with a carboxylic acid functionality.    -   It is able to form an oil like product with typical values as        specified below. Oil should be synthesised extracellularly and        physically separate to the top of the reaction media.

In one embodiment, the bacterium is Acetobacter lovaniensis FJ1.

Production of Carbon One Building Blocks (Formic Acid)

The micro-organism should reduce carbon dioxide to formic acid using ahydrogenase enzyme system. Hydrogen is oxidised to generate electrons toreduce carbon dioxide to formic acid as follows:

CO₂+H₂→HCOOH

This enzyme system functions in a cell free extract which impliesextracellular activity.

Assembly of Formic Acid into Short, Medium and Long Chain Hydrocarbons

The micro-organism should be capable of generating formic acid andforming more complex molecules by the stepwise addition of formic acid.High levels of formic acid are formed and a minimum level of 250 g perlitre of formic acid equivalents is maintained. This is where the levelof acidity is calculated as formic acid. Short chain aliphaticcarboxylic acids form first.

5 Day Culture 10 day culture Volatile Acid mg/l mg/l Acetic Acid (C2)152 619 Propionic Acid (C3) 406 1840 Butyric Acid (C4) 276 1220 ValericAcid (C5) 272 1250 Iso Butyric Acid (C4) <50 <50 Iso Valeric Acid (C5)<50 <50

The presence of C2, C3, C4 and C5 aliphatic carboxylic acids impliesstepwise addition of formic acid, since odd and even chain lengths arerepresented. Iso butyric and Iso valeric acid were either absent orbelow the level of detection demonstrating that straight chain aliphaticcarboxylic acids were produced. The amount of any given aliphaticcarboxylic acid increases with time.

The system is then held in equilibrium between the production of formicacid and the formation of larger molecules. At equilibrium, the quantityof oil generated (as measured by dry weight) is 5-10% of the acidity asmeasured as formic acid equivalents. Oil is then generated on acontinuous basis by the removal of an oily layer at the air liquidinterface and the addition of an equal volume of fresh media. The upperoily layer is pooled and concentrated.

The oil product consists of a mixture of carboxylic acids of varyingchain lengths. The length of chain is adjusted by the rate of removalfrom the primary culture. Chain length and proportions of moleculesgenerated are varied by adjusting the reaction conditions and the typeof equipment used. Types of reactors that can be used include but arenot limited to batch, continuous and semi-continuous. Reactor typesinclude but are not limited to closed reactors, open reactors, paddlestirred, circulated, and gas lifted.

Production of oil can be on a semi-continuous basis. The organism ismaintained in batch culture using a minimal media as detailed in thetable below. This is the “stock culture”.

NUTRIENT g/litre KH₂PO₄ 1.00 MgSO₄•7H₂O 0.10 CaCO₃ 0.10 CuSO₄ 0.01 FeCl₃0.01 MnCl₃ 0.01 MoCl₃ 0.01 ZnCl₃ 0.01

The pH of the media is adjusted to 4.0±0.2. The acidity in the stockculture is maintained at 20-25%, calculated as formic acid.

Before use in a reaction tank, the cell culture media is diluted 1/10 inwater and held in an intermediate tank. This is the working culture.

Oil is produced in reaction tanks including but not limited to the tanksystems described below. Sources of carbon dioxide include but are notlimited to atmospheric carbon dioxide, carbonate, bicarbonate ions,waste waters containing carbonate or bicarbonate ions, compressed gasand exhaust gasses containing carbon dioxide.

Tank System 1

The reaction tank is a long narrow tank (FIG. 2) constructed fromplastic or plastic coated material. Female connectors of a suitable sizeare set in the side of the tank to allow addition of media or removal ofproduct. Typical dimension would be 5 units long by 1 to 1.5 units wideand 0.5 to 1.0 units deep. A partition is set in the tank ⅕ from one endof the tank. This partition is ⅘ the height of the tank. Circulatingpumps with hoses are added to each partition. A sparge bar forre-circulation of contents can also be added to each partition.Compressed carbon dioxide can be additionally added to the reaction tankvia a gas cylinder and line. A heater or heater lamp may be positionedover the smaller of the two sections of the tank.

To initiate production of oil in the reaction tank, the larger sectionof the reaction vessel is filled with stock culture media. The media isthen circulated until the pH has increased to between 5 and 7 and oilseparates to the upper surface. The pump is then switched off.

Alternatively, to initiate production of oil in the reaction tank, thelarger section of the reaction vessel is filled with stock culture mediaand a small quantity of oil is added to act as a primer. The oil andstock culture is mixed by circulation until an emulsion forms.Additional carbon dioxide is pumped through the reaction mixture untilconversion of emulsion to oil has taken place. Conversion to oil ismeasured by the reduction of the level of water in the reaction mixture.

Oil may be removed from the upper surface by pulling a fat collector orboom across the upper surface, displacing the oil into the smaller ofthe two sections of the tank. The volume displaced is replaced by anequal volume of working culture media.

When the partition is full and the reaction is complete, the oil ispumped into an upright settling tank and allowed to stand. Ananti-microbial agent is added and the water allowed to settle out. Wateris removed through the lower valve.

Oil may be further processed by filtration or by removal of water tospecified levels using either demulsifying filters or drying agents.

In an alternative method, 2 litres of culture medium containing 20%bacteria is mixed with disinfectant to kill the bacteria prior tocommencing the production of oil. This culture medium contains theenzymes needed to convert carbon dioxide to formic acid and then toaliphatic carboxylic acids.

The culture medium containing the enzymes (and killed bacteria) is addedto a reaction tank which already contains 200 litres of waste oil. 50litres of water is then added each hour until the reaction tank containsabout 2000 litres of liquid. The bacterial enzymes only produce thealiphatic carboxylic acids when the reaction tank contains more oil thanwater. As the enzymes produce the aliphatic carboxylic acids (in theform of an oil), the level of oil increases. As oil is produced, thelevel of water is increased in the tank to displace the oil into a weir.The reaction tank is replenished at intervals with further water tosustain the production of oil.

The pH of the reaction tank is maintained at about 6.5 and thetemperature at about 15-20° C.

The reaction is paused at intervals to allow release of dissolved gasesand for the electron balance of the system to re-equilibrate.

Once the desired lengths of aliphatic carboxylic acids have beenproduced in the reaction tank, oil is removed from the upper surface bypulling a fat collector or boom across the upper surface, displacing theoil into a smaller section of the reaction tank. The viscosity of theoil gives an indication of the length of the aliphatic carboxylic acids.The oil is pumped into a separate tank and any residual water removed tostop any further reactions from taking place, thereby maintaining thelength of the aliphatic carboxylic acids at the desired length. Ananti-microbial agent is also added before the oil is filtered.

Tank System 2

A second option comprises an upright mixing tank with a conical bottom,recirculation via a pump, a thermostated immersion heater and a spargeto the bottom of the tank to introduce compressed carbon dioxide gas(FIG. 3). Stock enzyme solution, a small quantity of oil to act as aprimer and water are added to the tank. Optionally, a CO₂ sequesteringagent such as triethanol amine or PEG 300 can also be added. Further, amild oxidising agent (such as sodium hyperchlorite or sodium hydroxide)may be added to help initiate the reaction. The heater is set to atemperature of 35° C. and the contents mixed by circulation so as toform an emulsion. Carbon dioxide is introduced via the gas sparge at thebottom of the tank. The conversion of the oil-water emulsion to thealiphatic carboxylic acid oil is again followed by reduction in thelevel of water in the tank.

When the reaction is complete, the heat, circulation and gas flow areterminated and the contents allowed to settle. Any free water isdischarged through the bottom of the tank and the oil pumped to a secondsettling tank via 20 micron filter. The oil is allowed to further settleto remove residual water. Waste water is either re-circulated or can bedistilled to remove organic material as a light oil. (FIG. 3). Oil canbe combusted through a combined heat and power engine to generate heatand electricity. Exhaust emissions from the engine can then be recycledvia a scrubber unit consisting of any suitable carbon dioxidesequestering chemical, including but not limited to polyethylene glycol300 (PEG 300).

Oil may be further processed by filtration or by removal of water tospecified levels using either demulsifying filters or drying agents.

Tank system 1 and 2 differ in the time required to form oil product.Tank system 1 is a low energy consuming option but requires extendedreaction times and Tank system 2 is a more rapid technique but requiresa higher energy input.

The oil is tested to specified limits.

The oil may be processed further depending on the application for whichthe oil is intended. In some instances, the oil may be blended withanother oil, for example, in order to modify theproperties/characteristics of the other oil.

The Oil Product

An oil product is obtained with the following typical values:

SPECIFICATION ANALYSIS UNITS VALUE (SUGGESTED) Identification by IR —Aliphatic Aliphatic Carboxylic Carboxylic Acid Acid Ash m/m 0.030.025-0.050 Water Content m/m 2.7 0.05 max Flash Point ° C. 198 190 minViscosity @ 40° C. cSt 23.092 20-25 (50 max) Viscosity Index — 55 50 minDensity @ 20° C. Kg/l 938 920-950 Iodine Value mg I/100 g 90.6 85-95Acid Value mg KOH/g 147 140-160 Peroxide Value meg/kg 2.9 3 maxCalorific Value Gross MJ/kg 38.12 37 min

Alternatively, an oil product can be obtained with the following typicalvalues:

SPECIFICATION ANALYSIS UNITS VALUE (SUGGESTED) Identification by IR —Aliphatic Aliphatic Carboxylic Carboxylic Acid Acid Ash m/m 0.030.025-0.050 Flash Point ° C. 105 100 min Viscosity @ 40° C. cSt 50 maxDensity @ 15° C. Kg/l 0.853  0.85-0.882 Iodine Value mg I/100 g 90.685-95 Acid Value mg KOH/g 147 140-160 Peroxide Value meg/kg 2.9 3 maxOxidative Stability H 48+ 48 min Sulphur Content m/m 0.08 0.10 maxCalorific Value Gross MJ/kg 43.42 37 min Cetane Index 50 50 min Sedimentpotential % mass/mass <0.2 (typically 0.08)

Oil consists of aliphatic carboxylic acids:

The infra red fingerprint (FIG. 1) is typical of aliphatic carboxylicacids.

The oil reacts chemically as typical of a carboxylic acid.

The oil can used as short, medium or long feedstock.

The oil typically has a chain length of greater than 2 and less than 24.

The oil is typically mono unsaturated with an iodine value of 90-95 mg1/100 g.

The oil product undergoes the reactions of a carboxylic acid either as ashort, medium or long chain feed stock to yield other commerciallyuseful products. These reactions include but are not limited to:

a. Production of esters by reaction with alcohols using lipases, acidcatalysts or alkali catalysts.

b. Production of amides either via the ester intermediate or directlyvia the Schmidt reaction.

c. Production of carboxylate salts with bases including but notexclusively alkalai hydroxides, carbonates or hydroxyl carbonates.

d. Reduction to alcohols directly by hydrogenation or via the aldehydeintermediate using catalysts including but not exclusively NNdimethylchloroethylammonium chloride and lithium aluminium hydride.

e. Reduction to aldehydes either directly by hydrogenation or via anintermediate such as the acid halide emplying the Rosenmund reduction orthe thioester via the Fukuyama reduction.

f. Production of acid chlorides using reagents that include but notexclusively sulphur dichloride oxide, phosphorous (V) chloride andphosphorous trichloride

g. Decarboxylation of the carboxylic acid or acid salt eitherenzymatically with decarboxylases or with soda lime to generate theequivalent hydrocarbon.

h. Reduction size vi the Barbier Wieland degredation.

The oil is produced directly and not by the prior production of bio massand its subsequent processing.

The oil is not produced by fermentation.

The method of producing oil is “driven” in a novel way. The first stepof the process is the reduction of carbon dioxide to produce formicacid. Formic acid is a reducing agent (redox potential −0.25). The firststep of the reaction fixes carbon dioxide but also generates sufficientreducing equivalents to drive the next part of the process (chainbuilding). The use of an “in situ” chemical battery could be used todrive other redox reactions. Formic acid (reductant) then reducesavailable carboxylic acids (oxidant), adding one carbon to a growingchain and releasing oxygen as O₂. This circumvents the need to add areducing agent to drive the reaction, carbon and reducing equivalentsbeing sequestered in the final product.

Step 1—CO₂+H₂→HCOOH

Step 2—RCOOH+HCOOH→RCH₂.COOH+O₂

Initial steps in chain building are enhanced by the tendency of formicacid (and most short chain carboxylic acids) to form dimers in solution.

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1. A micro-organism comprising a hydrogenase enzyme system which is capable of converting carbon dioxide into formic acid and a second enzyme system which is capable of converting formic acid into aliphatic carboxylic acids having a chain length of five or more carbon atoms. 2-10. (canceled)
 11. The micro-organism of claim 1, wherein the micro-organism is the Acetobacter strain having accession number NCIMB
 41808. 12-18. (canceled)
 19. A method for producing aliphatic carboxylic acids, the method comprising producing aliphatic carboxylic acids using a medium comprising a hydrogenase enzyme system which is capable of converting carbon dioxide into formic acid and a second enzyme system which is capable of converting formic acid to aliphatic carboxylic acids having a chain length of five or more carbon atoms.
 20. The method of claim 19, wherein the medium comprises a micro-organism according to claim
 1. 21. The method of claim 19, wherein the reaction to produce the aliphatic carboxylic acids takes place at a pH of between about 3.0 and about 8.5.
 22. The method of claim 19, wherein the reaction to produce the aliphatic carboxylic acids takes place at a temperature of between about 5° C. and about 60° C.
 23. The method of claim 19, wherein carbon dioxide is the sole source of carbon.
 24. The method of claim 23, wherein carbon dioxide is bubbled through the medium.
 25. The method of claim 19, wherein the medium is an oil-water emulsion.
 26. The method of claim 19, wherein the medium comprises a carbon dioxide sequestering agent.
 27. The method of claim 19, wherein the medium comprises an oxidising agent.
 28. The method of claim 19, further comprising one or more of the following steps: 1) separating the aliphatic carboxylic acids; 2) filtering the aliphatic carboxylic acids; 3) blending the aliphatic carboxylic acids with a different fuel, preferably an oil fuel; 4) chemically modifying the carboxylic acids, for example, into ester, alcohols, ketones or aldehydes; and 5) distilling and separating certain fractions from the aliphatic carboxylic acids.
 29. A medium comprising a hydrogenase enzyme system which is capable of converting carbon dioxide into formic acid and a second enzyme system which is capable of converting formic acid to aliphatic carboxylic acids having a chain length of five or more carbon atoms.
 30. (canceled)
 31. Aliphatic carboxylic acids produced by the method of claim
 19. 32. (canceled)
 33. The aliphatic carboxylic acids of claim 31, wherein the aliphatic carboxylic acids have a chain length of between about 5 and about 24 carbon atoms.
 34. The aliphatic carboxylic acids of claim 33, wherein the aliphatic carboxylic acids are mono-unsaturated.
 35. The aliphatic carboxylic acids of claim 31, wherein the aliphatic carboxylic acids are in the form of an oil.
 36. The aliphatic carboxylic acids of claim 31, the aliphatic carboxylic acids having one or more of the following properties: Analysis Typical Value Ash g/100 g 0.025-0.050 Density @ 15° C. 0.8483-0.8720 Flash Point ° C. >100 Kinematic Viscosity cSt @ 20° C. 70-75 @ 30° C. 52-57 @ 40° C. 47-52 @ 50° C. 30-35 @ 60° C. 20-25 @ 70° C. 15-20 @ 80° C. 10-15 @ 90° C.  7-12 Viscosity Index 55 Water Content g/100 g 1 max (pref. <0.5) Acid Value mg KOH/g 140-160 Iodine Value mg I/100 g 85-95 Copper Strip Corrosion 1B Oxidative Stability H 48+ hours Sulphur % m/m <0.1 Peroxide Value meq/kg <3 Cetane Index ~50 Gross Calorific Value >37 Mj/kg Sediment potential <0.2 (typically 0.08)

37-45. (canceled)
 46. The method of claim 21, wherein the reaction to produce the aliphatic carboxylic acids takes place at a pH between about 6.0 and about 7.0.
 47. The method of claim 22, wherein the reaction to produce the aliphatic carboxylic acids takes place at a temperature of between about 15° C. and about 20° C. 